The present invention relates to C8-substituted purine nucleotide analogs and their use as inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases), and is particularly concerned with such compounds which provide effective and specific inhibition of NTPDases.
In 1971, results of extensive studies on neurotransmission, which was resistant to conventional adrenergic and cholinergic antagonists, led Burnstock to propose that the purine nucleotide ATP and/or the purine nucleoside adenosine, released at synaptic junctions, might mediate a non-adrenergic, non-cholinergic signalling (1-7). Burnstock also hypothesized that nerves released purines which interact on their target cells with purinergic receptors (or purinoceptors) for either ATP, or its breakdown product adenosine (8, 9). The putative ATP-selective receptors were termed P2-purinoceptors, whereas the adenosine receptors, were termed P1-purinoceptors (10). Soon, purinoceptors were identified, characterized, and localized in a variety of systems, organs, cells and cell extracts. At the beginning, purinoceptors were classified according to their pharmacological and physiological properties, but with the advent of molecular biology tools, genes encoding purinoreceptors were cloned and a new classification emerged (see 11 for a complete review). Extracellular ATP and ADP and its metabolite adenosine exert multiple effects through these purinoceptors. In the cardiovascular system, these compounds influence platelet aggregation, vascular tone, heart function and recruitment of blood cells involved in inflammatory processes (12-15). In the digestive system, it affects electrolyte secretion, gastrointestinal motility, stomach acid secretion and other secretions coming from accessory glands: parotid, liver and exocrine pancreas (16-20). Presence of purinoceptors in the immune system also support a role of extracellular purines and pyrimidines in the immune response (11, 21-25). Presence of these receptors in the central and peripheral nervous systems also supports a role in neurotransmission for these compounds (26-29). These localizations combined with the effects induced by the administration of nucleotides confirm the functions of these nucleotides and their metabolites.
A fundamental question is what determines extracellular concentrations of nucleosides and nucleotides in the extracellular compartment. Basically, these are five parameters involved: 1-Rate of release from the source (cell); 2-Rate of diffusion and size of the extracellular compartment; 3-Metabolism by ectonucleotidases; 4-Binding to proteins on the cell surface; and 5-Uptake by the cells (translocation or endocytosis). Ectoenzymes with ectonucleotidase activities often localized in proximity of the target cells are believed to play key roles as modulators of the purine or pyrimidine actions. Among the ectoenzymes which display ectonucleotidase activities, one finds alkaline phosphatase [EC 3.6.1.3] which is widely distributed in the different systems of the body, protein kinase reported in certain cell types, ecto-nucleotide pyrophosphatase/phosphohydrolase [EC n.d.] which converts nucleoside triphosphate into nucleoside monophosphate and 5xe2x80x2-nucleotidase [EC 3.1.3.5] which convert nucleoside monophosphate into nucleoside (30-34).
Ectonucleotidases, often located on the target cells, rapidly dephosphorylate the nucleotide into the corresponding nucleoside thereby ending the P2 stimulation and thereby inducing a P1 type stimulation (31, 33, 36). Quite often, the physiological response elicited by the nucleoside antagonizes the action induced by the corresponding nucleotide (adenosine vs ATP) (14-15). Adenosine is generally considered as a negative feedback modulator (retaliatory metabolite) of cell and organ energy demand and consumption. It interacts with P1 purinoceptors which comprise at least four subtypes A1, A2A, A2B and A3. first classified into those that inhibit (A1) and those that stimulate adenylate cyclase (A2) (11). They were later classified according to their pharmacological properties and they are now distinguished by their amino-acid sequences (11).
Once released, nucleotides and nucleosides diffuse in the extracellular space and reach their receptor on target cells. Many enzymes contribute to the extracellular metabolism of nucleotides including alkaline phosphatase, ectokinases and deaminases. Perhaps the most important ones are those that convert nucleotides and nucleosides. Many reports have described ecto-ATPase, ecto-ADPase, and ecto-5xe2x80x2-nucleotidase activities in a variety of tissues and cells. The latter was purified, characterized biochemically, and its encoding gene was defined (34). As for the conversion of ATP to ADP and AMP, up until recently, it was believed that two distinct ecto-enzymes were involved in the conversion of ATP to ADP, and ADP to AMP, i.e., ecto-ATPase and ecto-ADPase, respectively (30). The detection of the NTPDase at the surface of vascular cells has presented another alternative for the conversion of ATP to AMP at the cell surface (37). The identification of a mammalian ATP diphosphohydrolase or apyrase goes back to the early 1980s when LeBel et al. described an enzyme that could sequentially catalyse the hydrolysis of xcex3 and xcex2 phosphate residues of triphospho- and diphosphonucleosides (38). In a series of studies, the enzyme was purified, characterized, and identified as an ectoenzyme (39). A second isoform was identified, purified, and characterized in the bovine aorta (40) and placenta (41). Recent reports describing the homology between potato apyrase and human CD39, showing a comparison of bovine and porcine ATPDases, and the cloning and sequencing of the human ATPDase cDNA and reexpression of the human protein in COS cells, led to the demonstration that ATPDase isoform II and CD39 were the same protein (42-44).
Among many reported inhibitors of NTPDases, one finds analogs of purines, heavy metals, such as Cd2+ and Hg2+ (44, 46) and molecules belonging to the suramin family, Evans blue and also other types of molecules.
Purine analogs, such as xcex2, xcex3-MetATP, xcex2, xcex3-imido-ATP and ADPxcex2S, may be used to inhibit the NTPDase (47). These analogs share a common characteristic, that is they all bear a substituted group on the phosphate chain. Moreover, all these analogs are purinoceptor ligands. Other nucleotide analogs have also been reported as NTPDase inhibitors, mainly ARL67156 and PPADS. These analogs have been reported to inhibit ecto-ATPase activity (48-51). Finally, two other purine analogs have been reported as NTPDase inhibitors: fluorosulfonylbenzoyl adenosine (FSBA) and 2-thioether-AMP-S (46, 52). However, contrary to purine analogs, FSBA causes an irreversible NTPDase inhibition.
Many P2 antagonists related to suramin (53), reactive blue (54), reactive red (55), Evans blue (56), trypan blue (56) and small aromatic isothiocyanoto-sulphonates (57), have been reported to be ecto-nucleotidase inhibitors. Other molecules have been proposed as non-specific NTPDase inhibitors, such as sodium azide, sodium fluoride (46) and 9-amino-1,2,3,4-tetrahydroacridine or THA (58).
Based on the facts that (a) NTPDases play a major role in the regulation of purine nucleotide and nucleoside levels and (b) purine nucleotides and nucleosides are involved in and influence a number of biological processes, modulation of the activity of NTPDases may have significant effects on such biological processes. Therefore, there exists a need for effective inhibitors of NTPDases, to better modulate the activity of NTPDases, thus modulating the levels of purine nucleotides and nucleosides, which in turn results in the modulation of a variety of biological processes.
An aspect of the present invention is a C8-substituted purine nucleotide analog, wherein the analog is substituted at the C8 position with a substituent other than H.
A further aspect of the present invention is a composition comprising the above-mentioned analog in admixture with a suitable diluent or carrier.
Yet a further aspect of the present invention is a method for modulating the activity of an NTPDase enzyme comprising exposing the enzyme to the above-mentioned analog or composition.
In a preferred embodiment, the present invention provides a method for inhibiting the activity of an NTPDase enzyme comprising exposing the enzyme to the above-mentioned analog or composition.
Yet a further aspect of the present invention is a method for modulating the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in a biological system, comprising the step of introducing into said system the above-mentioned analog or composition.
Yet a further aspect of the present invention is a method for modulating the activity of a biological process in a biological system, wherein said process is affected by the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in said system, comprising the step of introducing into said system the above-mentioned analog or composition.
Yet a further aspect of the present invention is a use of the above-mentioned analog or composition for modulating the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in a biological system.
Yet a further aspect of the present invention is a use of the above-mentioned analog or composition for modulating the activity of a biological process in a biological system, wherein said process is affected by the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in said system.
Yet a further aspect of the present invention is a commercial package containing the above-mentioned analog or composition together with instructions for modulating the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in a biological system.
Yet a further aspect of the present invention is a commercial package containing the above-mentioned analog or composition together with instructions for modulating the activity of a biological process in a biological system, wherein said process is affected by the level of purine nucleotide(s) and/or nucleoside(s) and/or metabolite(s) or derivative(s) thereof in said system.